The aggressive cancer known as pancreatic ductal adenocarcinoma (PDAC) has a remarkably poor response to treatment, especially gemcitabine (GEM). As a member of the noncanonical IκB kinase family, inhibitor of nuclear factor kappa-B kinase subunit epsilon (IKBKE) is known to regulate tumor progression in multiple cancer types. However, its functional role in modulating chemosensitivity and its impact on cell death pathways in PDAC remain unclear.

We examined IKBKE expression in patient tumor tissues and publicly available databases and assessed its prognostic value to investigate its function in PDAC chemoresistance. Loss-of-function approaches, including shRNA knockdown and pharmacological inhibition using amlexanox, were employed in vivo and in vitro. Furthermore, cytotoxicity along with cell death patterns induced by GEM were assessed through flow cytometry, electron microscopy, and CCK-8 assays. Co-IP and GST pull-down assays were applied to determine whether IKBKE interacts with gasdermin E (GSDME), the executioner protein of pyroptosis. Moreover, in vitro kinase assays, phosphorylation mass spectrometry, and site-specific gain-/loss-of-function mutant functional experiments were conducted to investigate the specific pathways via which IKBKE affects GSDME-mediated pyroptosis. The therapeutic potential of IKBKE targeting was further validated using patient-derived tumor organoids and xenograft models.

IKBKE was significantly elevated in PDAC tissues and was closely linked to worse clinical outcomes. Functional studies of the signaling pathway showed that via the activation of the protein kinase B/glycogen synthase kinase-3β signaling pathway, IKBKE facilitates the aggressive phenotype of PDAC cells. IKBKE inhibition or silencing increased GEM sensitivity and caused caspase-3/GSDME-dependent pyroptosis in PDAC cells. Co-IP and GST pull-down analysis revealed IKBKE–GSDME interaction. Further in vitro cleavage and kinase assays, mass spectrometry analysis, and mutant functional studies showed that IKBKE inhibits pyroptosis by phosphorylating GSDME at Thr6, thereby hindering its cleavage by caspase-3. Treatment with amlexanox markedly suppressed tumor growth and synergized with GEM to enhance anti-tumor efficacy in organoid models by targeting IKBKE.

Our findings identify IKBKE as a key regulator of chemotherapy-induced pyroptosis in PDAC. Specifically, IKBKE phosphorylates GSDME at Thr6, which alters its conformation, thereby impeding caspase-3–mediated cleavage, ultimately contributing to GEM chemoresistance. IKBKE targeting reverses GEM resistance and enhances tumor immunogenic cell death via the caspase-3/GSDME pathway, supporting IKBKE inhibition as a promising therapeutic strategy for PDAC.